Virulence and resistance profiling of Staphylococcus aureus isolated from subclinical bovine mastitis in the Pakistani Pothohar region

Mastitis is considered one of the most widespread infectious disease of cattle and buffaloes, affecting dairy herds. The current study aimed to characterize the Staphylococcus aureus isolates recovered from subclinical mastitis animals in Pothohar region of the country. A total of 278 milk samples from 17 different dairy farms around two districts of the Pothohar region, Islamabad and Rawalpindi, were collected and screened for sub clinical mastitis using California Mastitis Test. Positive milk samples were processed for isolation of Staphylococcus aureus using mannitol salt agar. The recovered isolates were analyzed for their antimicrobial susceptibility and virulence genes using disc diffusion and PCR respectively. 62.2% samples were positive for subclinical mastitis and in total 70 Staphylococcus aureus isolates were recovered. 21% of these isolates were determined to be methicillin resistant, carrying the mecA gene. S. aureus isolates recovered during the study were resistant to all first line therapeutic antibiotics and in total 52% isolates were multidrug resistant. SCCmec typing revealed MRSA SCCmec types IV and V, indicating potential community-acquired MRSA (CA-MRSA) transmission. Virulence profiling revealed high prevalence of key genes associated with adhesion, toxin production, and immune evasion, such as hla, hlb, clfA, clfB and cap5. Furthermore, the Panton-Valentine leukocidin (PVL) toxin, that is often associated with recurrent skin and soft tissue infections, was present in 5.7% of isolates. In conclusion, the increased prevalence of MRSA in bovine mastitis is highlighted by this study, which also reveals a variety of virulence factors in S. aureus and emphasizes the significance of appropriate antibiotic therapy in combating this economically burdensome disease.


Study population and sample collection
A total of 17 peri-urban dairy farms from two districts of Pakistan, falling in the Pothohar region, namely Islamabad and Rawalpindi were included in the study.These dairy farms were small-scale to medium-scale holdings having the number of animals ranging from 15 to to 45 per dairy farm.Within each farm, the number of collected samples varies from 5 to 15 depending on the size of the herd.A total of 278 milk samples were collected from these dairy farms in a sterile falcon tube (15 mL) during January 2021 to October 2021.All the animals were apparently healthy and animals suffering from clinical mastitis with visible symptoms were excluded from study.Each milk sample was collected by pooling streaks from all of the four quarters of the teat per animal.Thus, a single animal was designated the sampling unit in this study in accordance with ARRIVE guidelines.Teats were disinfected with 70% ethanol solution before sample collection to discourage contamination with teat microflora.The study was approved by the COMSATS ethical review board (letter no: CIIT/Bio/ERB/2020/12) and animals were handled following the specified regulations and reported in accordance with the ARRIVE and STROBE-vet guidelines.Milk samples were analyzed for mastitis using the California Mastitis Test (CMT) as described earlier 18 .A trained veterinarian, previously briefed about the study, was engaged to collect samples and conduct the CMT test.The samples were transported to the Animal Health Program of Animal Sciences Institute, National Agricultural Research Centre, Islamabad while maintaining cold conditions.. Samples were stored at 4 °C till further processing.

Bacterial isolation and culture
Samples were enriched in Nutrient Broth (Oxoid, UK) [19][20][21] .A total of 9 ml of Nutrient broth was taken in a sterile universal bottle. 1 ml of milk sample was added in broth bottles and mixed.All milk samples were processed within 18 h after collection.The bottles were incubated overnight at 37 °C.Mannitol Salt Agar (MSA) (Oxoid, UK) was used to culture the samples.Isolates were next subjected to catalase test, coagulase test, and hemolysis for preliminary identification [22][23][24] .S. aureus was identified depending on the morphological properties observed on culture media and biochemical characteristics of isolate 25 .

Molecular identification of isolates
Bacterial DNA was extracted by modified boiling method.In this method, colonies from isolated bacterial cultures were used.A few colonies were immersed and suspended in 200µL lysis buffer (10 mM EDTA (2 mL) + 50 mM Tris HCL (4 mL) + 3% Triton (4 mL)) taken in Eppendorf tube and boiled for 10 min at 96 o C 26 .Immediately after boiling, the Eppendorf tubes are immersed in the ice bath for 5-10 min and centrifuged at 12,500 rpm for 5 min at room temperature.The DNA-containing supernatant is transferred to a new Eppendorf tube and processed for PCR or stored at − 20 °C for future use 27 .A 16S rRNA conserved gene was targeted in the confirmatory PCR for S. aureus.The thermal reaction mixture consisted of 2.5 µL of Taq buffer (Thermo Fisher Scientific, Waltham, USA), 1.5 µL of MgCl 2 ((Thermo Fisher Scientific, Waltham, USA),, 1µL of each forward and reverse primer (Macrogen, Korea), 0.5 µL of dNTPs (Thermo Fisher Scientific, Waltham, USA), 0.3 µL of Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, USA), and 15.2 µL of nuclease free water (Thermo Fisher Scientific, Waltham, USA),to make total volume of 22 µL.Total of 3 µL of template DNA was used in reaction.

Detection of mecA, SCCmec typing and virulence profiling
Methicillin resistance-conferring gene mecA is present in most MRSA strains.The mecA gene encodes an additional penicillin-binding protein 2a (PBP 2a), which mediates cell wall synthesis in the presence of β-lactam antibiotics which is located on a mobile genetic element known as staphylococcal cassette chromosome mec (SCCmec) 28,29 .Hospital-acquired MRSA carries SCCmec type I, II and III whereas community acquired MRSA carries SCCmec type IV and V.The protocols used by Boyle et al. and McClure-Warnier were followed for the identification of SCCmec types I to V 30 .The Panton-Valentine leukocidin (PVL) gene is a well-recognized, toxin producing virulence factor carried predominantly by community-acquired MRSA strains and is cytotoxic for the bovine neutrophils 31 .
We employed a multiplex PCR for detection of both mecA and Luk-PV (PVL) genes.Isolates possessing the mecA gene were classified as MRSA.In addition to PVL, the S. aureus isolates were screened for the presence of several additional virulence factors which included hemolysins, including alpha (α) and beta (β), encoded by hla and hlb genes respectively; nuc encoding an extracellular thermostable nuclease 32 and fnbA gene 33 .Furthermore, the isolates were screened for the presence of the bacterial cell wall component, capsular polysaccharide (CP) which helps in protection from phagocytic activity and enables immune system evasion.Cap5 and cap8 are most prevalent in S. aureus isolated from bovine and human infections 34 .Therefore their detection using multiplex PCR was performed as described earlier 35 .Similarly, the detection of fnbB, clfA, and clfB genes was performed using a multiplex PCR 33 .The Fishers exact test was used to compare the frequency of virulence genes among MRSA with that in MSSA isolates.

Antibiotic sensitivity test (AST)
Kirby Bauer Disc diffusion method was used to determine the antimicrobial susceptibility profile of S. aureus.Isolates were cultured on Mueller-Hinton agar plates (Oxoid, UK) after calibrating bacterial suspension to 0.5 MacFarlands standard.Twelve antibiotics (Oxoid, UK) including Penicillin G (10 µg), Amoxicillin (10 µg), Cephradine (30 µg), Cefoxitin (30 µg), Cefotaxime (30 µg), Ceftazidime (30 µg), Cefepime (30 µg), Oxytetracycline (30 µg), Ciprofloxacin (5 µg), Gentamicin (10 µg), Imipenem (10 µg), and Azithromycin (15 µg) were applied.Plates were incubated for 24 h and the zone of inhibition was determined.Results were interpreted based on CLSI guidelines (Clinical Laboratory Standard Institute (CLSI) 2018).Antibiotic selection was based on clinical relevance in veterinary and human health 36 .Typically, cefoxitin, oxacillin disk diffusion, and mannitol salt agar screen, is used to characterize methicillin resistance but the gold standard is the genotypic detection of the mecA gene.Isolates showing inhibition zone < 22 mm for cefoxitin were considered for mecA screening and those positive for the gene were identified as methicillin-resistant Staphylococcus aureus (MRSA).Additionally, the AST data was comprehensively analyzed to characterize S. aureus isolates as multidrug-resistant (MDR: resistant to ≥ one agent in ≥ 3 antimicrobial classes), extensively drug-resistant (XDR: resistant to one or more antibiotics in all tested classes, except 1 or 2 classes), as previously described 37 .

S. aureus prevalence in subclinical mastitis
A total of 278 milk samples were screened for subclinical mastitis by following the California Mastitis Test protocol as specified by the manufacturer.Of these, 173/278 (62%) animals were found positive for subclinical mastitis.
Out of 173 CMT positive samples 70 samples were culture positive for Staphylococcus aureus.Presumptive S. aureus colonies were sub-cultured and isolates were identified based on colony morphology and biochemical testing.The results showed a higher prevalence of CMT-positive samples and S. aureus isolates in Rawalpindi than in Islamabad.Distribution of CMT-positive cases and S. aureus isolation frequency also showed higher rates in cattle than buffalo.The district-wise and specie-wise CMT positive samples and S. aureus isolates are mentioned in Table 1.
16SrRNA conserved gene sequence for S. aureus was targeted in the confirmatory PCR for confirmation of isolate identity.In total, 70/173 (40%) isolates were confirmed as S. aureus.

MRSA identification and virulence characteristics
15 of the 70 (21%) isolates carried the mecA gene and were classified as MRSA.Among all Livestock Associated-MRSA strains, 33.3% of LA-MRSA carried SCCmec type IV whereas 13.3% of LA-MRSA carried SCCmec type V  www.nature.com/scientificreports/hence belonging to community acquired CA-MRSA which depicts that it was most likely transferred from farm handlers and workers who are in direct contact with livestock.Figure 2 depicts the overall analysis of SCCmec typing among LA-MRSA.Meanwhile, only 4/70 (5.7%) isolates possessed the PVL toxin gene.Virulence profiling showed the most common genes in MRSA isolates were hla, hlb, clfA and clfB.All the MRSA associated milk samples, especially the ones with clfA and clfB genes, were yellowish and had clots in it.Meanwhile, in MSSA isolates, the most common genes were hla, hlb, nuc, clfA and cap5.The frequency of clfa, clfb and LukPV genes among MRSA isolates was significantly higher than that for MSSA isolates (P < 0.05).All S. aureus isolates recovered in this study did not carry fnbA gene.Virulence genes distribution among MRSA and MSSA isolates is given in Table 3.

Discussion
Mastitis represents a significant challenge for the dairy industry in Pakistan, resulting in substantial production losses.Mastitis has been recognized as one of the most economically important diseases affecting dairy animals worldwide 38 .Meanwhile, subclinical mastitis also leads to decreased milk yield and poor milk quality and often goes undetected in herds leading to widespread dissemination.To determine the prevalence of  subclinical mastitis in dairy herds of Islamabad and Rawalpindi 278 pooled milk samples were collected from small to medium scale holdings in both districts.74% of milk samples were found positive for subclinical mastitis using the CMT from Rawalpindi as compared to 52% from Islamabad.Less incidence of sub clinical mastitis in Islamabad may be attributed to better hygienic conditions and literary status of farmers while in Rawalpindi high incidence of mastitis may be correlated with poor hygienic conditions at farm, as the chances of sub clinical mastitis increases due to poor animal hygiene 39,40 .In our study, overall subclinical mastitis prevalence was found to be 62%.This is higher than the prevalence rates reported in earlier studies 5,41 , and corresponds with high prevalence of subclinical mastitis reported earlier from the Pothohar region which includes Islamabad, Rawalpindi, Attock, Jehlum, and Chakwal districts 42 .These results depict the growing burden of subclinical mastitis within dairy animals in this region.Similar to previous reports we also found high prevalence (40%) of S. aureus in subclinical mastitis milk samples underlining its importance as the causative agent of bovine mastitis 5,7,41,43 .Methicillin resistant Staphylococcus was first confirmed as the causative agent of mastitis in dairy cattle in 1972 44 .The presence of MRSA in bovine mastitis poses a significant risk to farmworkers, including veterinarians, and can lead to severe intra-herd infections 45,46 .Although previous studies have reported a low prevalence of MRSA, ranging up to 4.3%, in Asia, a relatively higher prevalence of up to 6.47% has also been documented 47 .In our study we observed a much higher MRSA prevalence (21%) amongst S. aureus isolates from cases of bovine subclinical mastitis.Similarly, a high prevalence of MRSA (19.6%) was reported in another province of   www.nature.com/scientificreports/Pakistan, Khyber Pakhtunkhwa, highlighting the increasing prevalence of MRSA as a causative agent of bovine mastitis 7 .This trend could be attributed to the neglect of cleanliness practices during milking by milkers and farm management.. MRSA prevalence is linked to farm population, farming techniques, disinfectants used, and animal commerce 48 .Our results also showed that the majority of the MRSA isolates carried SCCmec type IV, while 13% of the isolates carried mec type V, which is associated with community acquired MRSA.The presence of CA-MRSA particularly mec (SCCmec) types IV and IVa has also been reported from Korea amongst mastitis affected dairy cattle 49 .The carriage of mec type V is often associated with CA-MRSA, suggesting the potential for transmission through farmer handling practices.S. aureus produces several virulence factors that help in its pathogenesis, resulting in mastitis 16 .There are two major classes of these virulence factors: surface associated structural components and secreted virulence factors, which help S. aureus evade host immune responses and cause chronic infection 50 .
The Panton-Valentine leukocidin (PVL) gene is a common, well-recognized, toxin-producing virulence factor of S. aureus.PVL is most cytotoxic for bovine neutrophils 31 .This toxin is also commonly associated with the methicillin-resistant S. aureus 51 .The presence of the PVL gene in methicillin-resistant S. aureus from other animals has been previously reported.In this study.All the PVL positive isolates were also positive for the mecA.In total, 5.7% of isolates tested positive for Luk-PV indicating the presence of PVL toxin with a higher occurrence in MRSA than MSSA, which is in accordance with previous reports of PVL gene occurrence amongst S. aureus and MRSA bovine isolates respectively 52,53 .
Haveri et al. ( 2008) presented that fnbA and fnbB genes coexist in the predominant PFGE types of S. aureus strains isolated from bovine intramammary infections 54 .Meanwhile, fnbB is more common than fnbA in S. aureus isolates from subclinical mastitis 55 .Our study also depicted a higher prevalence of fnbB, while fnbA was not detected among our isolates.The ability of adhesion to fibronectin is important during several steps of the disease process because fibronectin is a ubiquitous host protein present in soluble form in the blood and in fibrillar form in cellular matrices 56 .
The ability of S. aureus to bind to fibrinogen and fibrin is believed to be an important factor in the initiation of foreign-body and wound infections 57 .A receptor for fibrinogen called the clumping factor (ClfA) is located on the surface of S. aureus cells.The affinity is very high, and clumping occurs in low concentrations of fibrinogen 58,59 .Previously, the cell wall-anchored protein clumping factor B (ClfB) has been demonstrated to play a crucial role in facilitating nasal colonization of S. aureus.This occurs through high-affinity interactions with the cornified envelope within the anterior nares of humans.. ClfB exerts its effect in the early stage of infection, and its interaction with loricrin appears to play a role during pathogenesis and making this a vital virulence factor for vaccine formulation 60 .Our data shows the prevalence of fibrinogen-associated clumping factors clfA and clfB to be 55% and 52%, respectively, which is similar to previous reports 60 .
The capsular polysaccharide or capsule is a cell wall bacterial component that provides protection to bacterial cell against the phagocytic activity of immune cells.In previous studies the cap5 gene frequency was much higher (42-70%) than cap8 (6.6%) in bovine subclinical mastitis isolates 61,62 .In this study cap5 was detected among 45% and cap8 was detected in 8.5% of bovine subclinical mastitis isolates.
Extra cellular thermostable nuclease (nuc gene) is a common enterotoxin and virulence factor produced by S. aureus, which helps in evasion of immune cells of host 63 .In a recent study 31% of isolates had the nuc gene 64 .In this study nuc gene was detected and confirmed in 71% of S. aureus isolates.
Hla and hlb encode for hemolysin toxins and were detected in 82% and 67% of bovine subclinical mastitis isolates respectively.Similar prevalence has been reported from China and Egypt 15,65,66 .Hla and Hlb have been also reported as the most common toxin genes among S. aureus isolates in Pakistan 67 .Hemolysins, attack cell membranes, cause platelet damage, destruction of lysosome, necrosis, and ischemia.Hla (α hemolysin) is a beta-barrel pore-forming toxin that disrupts the cell membrane causing irreversible osmotic changes, resulting in cell-death by apoptosis 68 .Meanwhile Beta hemolysin (Hlb) is encoded by a lysogenic bacteriophage; in itself, it cannot destroy most cell types, but it exposes vulnerable cells to other lytic proteins, such as Hla and leukocidins like PVL 69 .
Antimicrobial resistance is fast becoming a major health threat.Multidrug resistant S. aureus infections are becoming common in recent times.It must be noted that failure to treat chronic S. aureus infection can lead to the bacterium penetrating the mammary gland tissue forming an abscess and eventual scar tissue formation 70 .In the current study we observed highest number of resistant isolates against the class penicillin (penicillin g and amoxicillin) as reported earlier from Pakistan 5,12 as well as globally [71][72][73] .Subclinical bovine mastitis S. aureus isolates in this study also showed very high resistance against 1st generation cephalosporins (cephradine) and moderate resistance against the 3rd generation cephalosporins (cefotaxime), as has been observed earlier 11 .
Macrolide (azithromycin) resistance was also observed among 55% of S. aureus isolates asreported previously from the region 12 .
On the other hand, all the S. aureus isolates were sensitive towards other antibiotic classes including 2 nd and 4th generation cephalosporins (cefoxitin & cefepime), tetracyclines (oxytetracycline), fluoroquinolones (ciprofloxacin), aminoglycosides (Gentamicin), and carbapenem (imipenem).These results are consistent with previous observations 11 and present alternatives for therapy.Notably, gentamicin and enrofloxacin antibiotics are listed in the approved list of antimicrobial agents approved for veterinary medicine by the World Health Organization and World Organization for Animal Health 74 .
The use of antibiotics for the treatment of mastitis helps to a large extent in avoiding economic losses from the disease.Most of the S. aureus strains are normally sensitive to majority of antibiotics but antibiotic resistance is becoming a problem, so it is advisable to perform antibiotic sensitivity test to minimize the hazard of drug resistance and to avoid economic loss on treatment.In this study almost 52% of the isolates were found to be multidrug resistant.These figures are alarming and reflect the unsupervised usage of antibiotics in veterinary practices.

Conclusions
Overall prevalence of sub-clinical mastitis was high in Pothohar region of the country with a high frequency of S. aureus isolation from the affected teat.Virulence profiling of S. aureus isolates showed high prevalence of hemolysins hla, hlb, and factors associated with host immune evasion.Meanwhile the leucocidin, PVL was present in few isolates that also carried the methicillin resistance gene mecA.We report high prevalence of MRSA compared to previous reports indicating a possible selection pressure for mecA and PVL positive isolates.Antibiotics sensitivity test shows that all the strains of S. aureus were sensitive to ciprofloxacin, gentamycin, and oxytetracycline.Managers of dairy herds should adopt good milking technique, to limit bacterial spread amongst dairy animals.

Figure 1 .
Figure 1.Map showing milk sampling locations in Pothohar Region with MRSA & MSSA distribution.

Table 1 .
Distribution of CMT positive samples and S. aureus isolation frequency amongst various bovine species in two districts of Pothohar region.

Table 4 .
Common antibiotic susceptibility and resistance patterns observed in S. aureus isolates.